The biosynthesis of the myelin proteolipid (PLP) and other acylated brain proteins will be studies in several experimental systems. Amino acid and palmitic acid incorporation into PLP will be analyzed in rat brain slices, in quaking mouse brain slices and in primary cultures of fetal rat brain. These experimental systems provide different approaches to the study of PLP acylation. Since brain slices are close to the in vivo system, we will be able to characterize the membranes that are involved in vivo in PLP processing and acylation. In the quaking mouse mutant, PLP is synthesized but is not inserted into myelin. Investigation of PLP processing in this mutant may, therefore, provide insight into the regulation of myelin formation. The primary cultures are important because: 1) they can be experimentally manipulated to a greater extend than can brain slices and 2) oligodendrocytes are apparently programmed to make myelin constituents in culture in the absence of neurons, although they do not form normal multilamellar myelin. Thus, PLP-containing membranes isolated from these cultures will be somewhat different from the myelin-forming memebranes in vivo. New information about the stages of PLP processing and myelin formation will be obtained from these cultures. Subcellular membranes that contain 1) PLP prior to acylation and 2) the acylating enzyme for PLP will be purified from both brain slices and cultured cells. PLP samples will be analyzed by both chloroform-methanol solubility and immunoprecipitation. Two other brain proteins have been demonstrated to be acylated with H3-palmitic acid. The acylation of these proteins will be investigated to identify the cell type and subcellular membrane in which thay are found and their site of acylation. An important question will be whether the same enzyme acylates these proteins and PLP and what regulates these acylations. Brain proteins that contain myristic acid will also be studied. A number of proteins may be found with covalently bound myristic acid, including pp60src, a protein found in other cell systems to contain myristic acid. The enzyme(s) involved in both palmitic acid and myristic acid acylation of proteins will be studies after establishing a cell-free protein acylation system with brain membranes. The proposed studies will provide new insight into the synthesis and assembly of brain membranes, in particular myelin, and may ultimately shed some light on the mechanisms of myelin disorders such as multiple sclerosis or metachromatic leukodystrophy.